simultaneous-measurement-superoxidehydrogen-peroxide-nadh-production

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Memorial University of Newfoundland

Pyruvate dehydrogenase (Pdh) and 2-oxoglutarate dehydrogenase (Ogdh) are vital for Krebs cycle metabolism and sources of reactive oxygen species (ROS). O2(·-)/H2O2 formation by Pdh and Ogdh from porcine heart were compared when operating under forward or reverse electron transfer conditions. Comparisons were also conducted with liver and cardiac mitochondria. During reverse electron transfer (RET) from NADH, purified Ogdh generated ~3-3.5× more O2(·-)/H2O2 in comparison to Pdh when metabolizing 0.5-10µM NADH. Under forward electron transfer (FET) conditions Ogdh generated ~2-4× more O2(·-)/H2O2 than Pdh. In both liver and cardiac mitochondria, Ogdh displayed significantly higher rates of ROS formation when compared to Pdh. Ogdh was also a significant source of ROS in liver mitochondria metabolizing 50µM and 500µM pyruvate or succinate. Finally, we also observed that DTT directly stimulated O2(·-)/H2O2 formation by purified Pdh and Ogdh and in cardiac or liver mitochondria in the absence of substrates and cofactors. Taken together, Ogdh is a more potent source of ROS than Pdh in liver and cardiac tissue. Ogdh is also an important ROS generator regardless of whether pyruvate or succinate serve as the sole source of carbon. Our observations provide insight into the ROS generating capacity of either complex in cardiac and liver tissue. The evidence presented herein also indicates DTT, a reductant that is routinely added to biological samples, should be avoided when assessing mitochondrial O2(·-)/H2O2 production.

2-Oxoglutarate dehydrogenase is a more significant source of O2(·-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.

Here, we report that choline and dimethylglycine can stimulate reactive oxygen species (ROS) production in liver mitochondria. Choline stimulated O˙/HOformation at a concentration of 5 μm. We also observed that Complex II and III inhibitors, atpenin A5 and myxothiazol, collectively induced a 95% decrease in O˙/HOproduction indicating both sites serve as the main sources of ROS during choline oxidation. Dimethylglycine, an intermediate of choline oxidation, was a more effective ROS generator. Rates of production were ~ 43% higher than choline-mediated O˙/HOproduction. The main site for dimethylglycine-mediated ROS production was via reverse electron transfer to Complex I. Our results demonstrate that metabolism of essential metabolites involved in methionine and folic acid biosynthesis can stimulate mitochondrial ROS production.

Choline and dimethylglycine produce superoxide/hydrogen peroxide from the electron transport chain in liver mitochondria.

Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O/ HOemission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O/ HOproduction by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NADlowering O/ HOproduction. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O/ HOproduction in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.

Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

Marisa O'Brien

Department of Biochemistry

Memorial University of Newfoundland

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Marisa O'Brien

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry

Memorial University of Newfoundland

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Department of Biochemistry